Spatial analysis of transcript and protein levels in skeletal muscle

Area of Science:

• Molecular biology
• Anatomy
• Biochemistry

Background:

• Skeletal muscle was historically viewed as a uniform tissue.
• Recent spatial analyses reveal significant regional differences in gene expression within skeletal muscle.
• These findings challenge the traditional understanding of muscle homogeneity.

Purpose of the Study:

• To present a detailed protocol for the spatial analysis of both transcript and protein levels in murine skeletal muscle.
• To provide researchers with a reproducible method for investigating regional gene expression in muscle tissue.
• To establish a foundation for further studies into the functional implications of muscle heterogeneity.

Main Methods:

• Dissection of the tibialis anterior muscle in mice.
• Formaldehyde fixation and tissue sectioning using a tissue chopper.
• Hybridization Chain Reaction (HCR) for transcript detection and amplification.
• Immunostaining for protein localization.
• Tissue clearing and advanced imaging techniques.

Main Results:

• The protocol enables simultaneous spatial analysis of gene and protein expression within skeletal muscle.
• Demonstrates the feasibility of high-resolution mapping of molecular markers across muscle regions.
• Highlights the successful application of HCR and immunostaining in cleared murine muscle tissue.

Conclusions:

• The presented protocol offers a robust method for spatial transcriptomic and proteomic analysis in skeletal muscle.
• This technique facilitates the investigation of tissue heterogeneity at a molecular level.
• The protocol's adaptability makes it valuable for diverse biological tissues beyond skeletal muscle.